We have recently shown that human semen contains trillions of extracellular vesicles (SEV) carrying small extracellular RNA (exRNA) molecules that may regulate the immune system (Nucleic Acids Research, 42:7290-7304, 2014). In unpublished studies, we have found that SEV suppress adaptive T cell responses, most likely by affecting antigen presentation. SEV also associate with HIV virions in vitro and in our hands enhance HIV susceptibility, although another group recently reported the opposite. Ultimately, SEV likely impact HIV infection, both by directly interacting with HIV and its target cells, and by compromising protective T cell immunity against HIV. Opioids too affect the immune system, although the exact mechanisms and functional outcomes are not well- understood. Heroin abuse leads to an immunocompromised state and increases HIV susceptibility, but the behavioral and biological causes for this are difficult to untangle. Heroin addiction is often treated with long- term methadone or buprenorphine medication, two opioids whose immunological properties and effects on HIV infection also remain unclear. Thus, the interplay of extracellular vesicles in semen and opioid use may have important consequences for HIV infection and the immune system, and this proposal will investigate these connections. We will determine the effects of SEV on HIV infection in a vaginal explant model and dissect the nature and extent of SEV- mediated inhibition of adaptive T cell responses, including to HIV vaccination. We will perform comparative studies of HIV susceptibility, adaptive T cell responses and SEV biology (exRNA composition and function) in blood and semen samples from normal donors, as well as heroin users before and three months after starting assisted methadone or buprenorphine treatment. The three opioids will also be tested in culture, because expression of the Opioid Receptor-Like 1 receptor on vaginal leukocytes (our data), and reports of other non- opioid receptors such as Toll-like receptor 4, indicate that opioids could directly affect leukocyt biology. SEV and opioid interactions will be addressed in combinatorial experiments. These studies will deepen our understanding of how opioids and extracellular vesicles in semen, alone or in conjunction, impact sexual HIV transmission. Knowledge of these effects will help to improve HIV vaccination outcomes, focus other HIV prevention strategies, and provide new information on the clinical benefits of methadone versus buprenorphine substitution therapy.